One of the main difficulties in understanding the mechanisms of resistance to anti-HER2 agents is the concomitant treatment with chemotherapy. In this proposal, we took advantage of unique samples obtained from a patient who first achieved a pathological complete response to the anti- HER2 antibody trastuzumab but then relapsed in a distant lymph node overtime. Deep sequencing of these specimens revealed that the acquisition of drug resistance coincided with the appearance of a previously unreported mutation in the Akt 3 gene. This result is imputable entirely to the pharmacological pressure of trastuzumab as this patient did not receive any other concomitant therapies (including chemotherapy). The objective of this study is to investigate whether the acquisition of the newly identified Akt 3 mutation, R247C, leads to resistance to anti-HER2 therapy in HER2 positive breast cancer. We plan to investigate the prevalence of this mutation (or other putatively activating mutations of th Akt genes) in samples from patients who are or became refractory to anti-HER2 therapy. Moreover, we plan to study the intrinsic kinase activity of Akt 3 R247C and consequent acquisition of the resistance phenotype. Finally, we will assess whether catalytic inhibition of Akt, alone or in combination with anti-HER2 therapy, is efficacious in limiting proliferation and tumor growth in cells/tumors bearing the Akt 3 R247C mutation. We have already collected more than 20 paired biopsies (pre- and post-treatment) from HER2 positive breast cancer patients who initially responded to either trastuzumab or lapatinib (a small molecule tyrosine kinase inhibitor of HER2) but eventually progressed to therapy overtime. The analysis of these specimens by IMPACT, a platform that allows to exome sequence ~300 cancer- related genes, will reveal how many of these patients have acquired a mutation in the Akt genes or in other genes related to the PI3K/Akt/mTOR pathway. Since the R247C mutation of Akt 3 was never reported, we aim to study its potential role in activating the enzyme and confer resistance to anti-HER2 agents. Akt 3 R247C will be expressed in HER2 positive cells sensitive to trastuzumab and lapatinib. In cell culture, we plan to perform long term viability assays, both in 2D and 3D culture, to study the capacity of these stable clones to escape the inhibitory activity of these agents. A similar approach will be taken i vivo, where BT474 cells (tumorigenic) expressing both wild-type and mutated Akt 3 will be injected in nude mice to form xenografts that will be treated with anti-HER2 agents, alone or in combination. Finally, we will assess the efficacy of catalytic Akt inhibition in re-sensitize cells stably expressing Akt 3 R247C to anti-HER2 therapy. In particular, we will monitor tumor growth inhibition in response to the treatments and perform pharmacodynamics studies to confirm inhibition of the pathways.